Rarity of human T helper 17 cells is due to retinoic acid orphan receptor-dependent mechanisms that limit their expansion.

The reason why CD4(+) T helper 17 (Th17) cells, despite their well-known pathogenic role in chronic inflammatory disorders, are very rare in the inflammatory sites remains unclear. We demonstrate that human Th17 cells exhibit low ability to proliferate and to produce the T cell growth factor interleukin-2 (IL-2), in response to combined CD3 and CD28 stimulation. This was due to the upregulated expression of IL-4-induced gene 1 (IL4I1) mRNA, a secreted L-phenylalanine oxidase, which associated with a decrease in CD3ζ chain expression and consequent abnormalities in the molecular pathway that allows IL-2 production and cell proliferation. High IL4I1 mRNA expression was detectable in Th17 cell precursors and was strictly dependent on Th17 cell master gene, the retinoid acid related orphan receptor (RORC). Th17 cells also exhibited RORC-dependent CD28 hyperexpression and the ability to produce IL-17A after CD28 stimulation without CD3 triggering. Our findings suggest that the rarity of human Th17 cells in inflamed tissues results from RORC-dependent mechanisms limiting their expansion.

Therapeutic Efficacy of Autologous Non-Mobilized Enriched Circulating Endothelial Progenitors in Patients With Critical Limb Ischemia　- The SCELTA Trial.

BACKGROUND: The therapeutic efficacy of bone marrow mononuclear cells (BM-MNC) autotransplantation in critical limb ischemia (CLI) has been reported. Variable proportions of circulating monocytes express low levels of CD34 (CD14+CD34lowcells) and behave in vitro as endothelial progenitor cells (EPCs). The aim of the present randomized clinical trial was to compare the safety and therapeutic effects of enriched circulating EPCs (ECEPCs) with BM-MNC administration.Methods and Results:ECEPCs (obtained from non-mobilized peripheral blood by immunomagnetic selection of CD14+and CD34+cells) or BM-MNC were injected into the gastrocnemius of the affected limb in 23 and 17 patients, respectively. After a mean of 25.2±18.6-month follow-up, both groups showed significant and progressive improvement in muscle perfusion (primary endpoint), rest pain, consumption of analgesics, pain-free walking distance, wound healing, quality of life, ankle-brachial index, toe-brachial index, and transcutaneous PO2. In ECEPC-treated patients, there was a positive correlation between injected CD14+CD34lowcell counts and the increase in muscle perfusion. The safety profile was comparable between the ECEPC and BM-MNC treatment arms. In both groups, the number of deaths and major amputations was lower compared with eligible untreated patients and historical reference patients. CONCLUSIONS: This study supports previous trials showing the efficacy of BM-MNC autotransplantation in CLI patients and demonstrates comparable therapeutic efficacy between BM-MNC and EPEPCs.

Demethylation of the RORC2 and IL17A in human CD4+ T lymphocytes defines Th17 origin of nonclassic Th1 cells.

Th17-derived Th1 lymphocytes, termed nonclassic, differ from classic Th1 cells because of the presence of retinoic acid orphan receptor (ROR)C2 and the surface expression of CD161 and CCR6. We demonstrate in this article that nonclassic Th1 cells, like Th17 cells, have a marked RORC2 and IL17A demethylation, whereas classic Th1 cells exhibit a complete methylation of these genes. The analysis of RORC2 DNA methylation in the CD4(+)CD161(+) and CD4(+)CD161(-) naive Th subsets from umbilical cord blood surprisingly revealed comparable hypermethylation levels. PCR analysis at the single-cell level revealed that RORC2 mRNA was expressed by none of the CD4(+)CD161(-) and present only in a minority of CD4(+)CD161(+) naive Th cells. These findings provide two important novel observations on the physiology of human Th17 cells: 1) they confirm at the epigenetic level the origin of nonclassic Th1 cells from Th17 cells, also identifying in the RORC2 and IL17A methylation status a novel tool for their distinction from classic Th1 cells, and 2) they demonstrate that RORC2-expressing cells are only a minority in the subset of CD4(+)CD161(+) naive Th cells, which are known to contain all Th17 cell precursors.

IL-4-induced gene 1 maintains high Tob1 expression that contributes to TCR unresponsiveness in human T helper 17 cells.

Human Th17 cells have a limited proliferative capacity compared to other T-cell subsets. We have shown that human Th17 cells display impaired IL-2 production due to IL-4-induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B-cell traslocation gene) antiproliferative protein family, which prevents cell-cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR-activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)-related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR-mediated expansion not only by blocking the molecular pathway involved in the activation of the IL-2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.

Distinctive features of classic and nonclassic (Th17 derived) human Th1 cells.

T helper17 (Th17) lymphocytes represent a third arm of the CD4(+) T-cell effector responses, in addition to Th1 and Th2 cells. Th17 cells have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in inflammatory sites. In humans, Th1 cells derived from Th17 cells express CD161, whereas classic Th1 cells do not; these Th17-derived Th1 cells have been termed nonclassic Th1 cells. In this study, we examined similarities and differences between classic and nonclassic human Th1 cells by assessing a panel of T-cell clones, as well as CD161(+) or CD161(-) CD4(+) T cells derived ex vivo from the circulation of healthy subjects or the synovial fluid of patients with juvenile idiopathic arthritis. The results show that nonclassic Th1 cells can be identified based on CD161 expression, as well as the consistent expression of retinoic acid orphan receptor C, IL-17 receptor E, CCR6, and IL-4-induced gene 1, which are all virtually absent in classic Th1 cells. The possibility to distinguish these two-cell subsets by using such a panel of markers may allow the opportunity to better establish the respective pathogenic roles of classic and nonclassic (Th17 derived) Th1 cells in different chronic inflammatory disorders.

CD4+CD161+ T lymphocytes infiltrate Crohn's disease-associated perianal fistulas and are reduced by anti-TNF-α local therapy.

BACKGROUND: Crohn's disease (CD) is an idiopathic inflammatory bowel disease (IBD) in the pathogenesis of which both Th1 and Th17 lymphocytes have been described as being involved. The NK-associated molecule CD161 has recently been described as a marker of IL-17-producing lymphocytes. In this work we assessed the presence and the functional features of CD161 T helper lymphocytes infiltrating CD-associated perianal fistulas, both before and after inoculation of anti-TNF-α mAbs along the fistula. METHODS: In a group of 9 CD patients with fistulizing perianal disease, we evaluated phenotypic and functional features of T cells recovered from the fistula, comparing them with peripheral blood (PB) T lymphocytes. Moreover, the effects anti-TNF-α mAbs injections along the fistula in terms of ability to reduce the inflammatory infiltrate and to determine fistula disappearance were assessed. RESULTS: In CD patients with fistulizing disease there is an accumulation of CD161+ T helper lymphocytes, with higher frequencies of Th1, Th17 and Th17/Th1 cells in the fistula than in PB. Local anti-TNF-α administration is associated with fistula resolution in the majority of patients with disappearance of infiltrating T lymphocytes, without any systemic effect in circulating effector T cells. CONCLUSIONS: These findings suggest that CD4+CD161+ T cells with Th17, Th17/Th1 and Th1 phenotype accumulate in CD perianal fistulas, and indicate local anti-TNF-α mAbs administration along the fistula as a promising tool for the treatment of these patients.

Evidence of the transient nature of the Th17 phenotype of CD4+CD161+ T cells in the synovial fluid of patients with juvenile idiopathic arthritis.

OBJECTIVE: To investigate the phenotype and function of CD4+ T cells in synovial fluid (SF) from the affected joints of children with oligoarticular-onset juvenile idiopathic arthritis (JIA), and to establish a possible link with disease activity. METHODS: CD4+ T cells were obtained from the peripheral blood (PB) and SF of 23 children with oligoarticular-onset JIA, as well as from the PB of 15 healthy children. The cells were analyzed for the expression of CXCR3, CCR6, and CD161 and for the production of interferon-γ and interleukin-17A (IL-17A). Spectratyping and clonotype analyses were performed to assess different T cell subsets. RESULTS: The numbers of CD4+CD161+ cells showing either the Th1 or the Th17/Th1 phenotype were higher in the SF than in the PB of children with JIA. The few Th17 cells from JIA SF underwent a spontaneous shift to the Th1 phenotype in vitro, whereas Th17 cells from the PB of healthy children shifted only in the presence of JIA SF; this effect was neutralized by antibody blockade of IL-12 activity. Spectratyping and clonotype analyses showed a similar skewing of the T cell receptor V(ß) repertoire in both CD161+ Th17 cells and CD161+ Th1 cells derived from the SF of the same JIA patient. The frequencies of CD4+CD161+ cells, particularly the Th17/Th1 cells, in the JIA SF positively correlated with the erythrocyte sedimentation rate and levels of C-reactive protein. CONCLUSION: These findings suggest that a shifting of CD4+CD161+ T cells from Th17 to the Th17/Th1 or Th1 phenotype can occur in the SF of children with oligoarticular-onset JIA, and indicate that the accumulation of these cells is correlated with parameters of inflammation. Thus, the results support the hypothesis that these cells may play a role in JIA disease activity.

CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC.

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL-23 receptor (IL-23R), CCR6, CD161, and the transcription factor retinoic acid-related orphan receptor C (RORC), and originate from a CD161(+)CD4(+) naïve T-cell precursor in response to the combined activity of IL-1ß and IL-23. We show here that not only CD4(+)TCRαß(+), but also CD8(+)TCRαß(+), CD4(-)CD8(-) TCRαß(+), and CD4(-)CD8(-) TCRγδ(+) circulating lymphocytes that produce IL-17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8(+) and CD4(-)CD8(-) umbilical cord blood T cells that already express RORC and IL-23R mRNA and that can be induced to differentiate into IL-17-producing cells in the presence of IL-1ß and IL-23. Finally, we provide evidence that umbilical cord blood naïve CD4(+)CD161(-) T cells, upon lentivirus-mediated transduction with RORC2 can acquire the ability to express IL-23R, IL-1RI, and CD161, as well as to produce IL-17. Taken together, these data allow to conclude that T-cell subsets able to produce IL-17, as well as precursors of IL-17-producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2-dependent.

Frequency of regulatory T cells in peripheral blood and in tumour-infiltrating lymphocytes correlates with poor prognosis in renal cell carcinoma.

OBJECTIVE: • To compare the frequency of T regulatory cells (Tregs) in peripheral blood of patients (pPB) affected by renal cell carcinoma (RCC) both with the frequency of Tregs found in PB of healthy donors (hPB) and that of Tregs present in tumour infiltrating lymphocytes (TILs). To verify in vitro the inhibitory activity of tumour isolated Tregs on the effector T cells and, finally, to assess the prognostic role of Treg frequency determination. PATIENTS AND METHODS: • Treg frequency in hPB, pPB and TILs was evaluated in 30 patients and 20 healthy controls by measuring both membrane-CD25 and intracytoplasmic-Foxp3 expression by flow cytometry. • Treg inhibitory activity was evaluated by an in vitro proliferation assay performed on total, CD25-depleted mononuclear cells (MNC) and CD25-depleted MNC cultured in the presence of purified CD25(+) Tregs. • Finally, Treg frequency in pPB and TIL were correlated with conventional prognostic factors and scores of University of California Los Angeles and Kattan predictive models. RESULTS: • Treg frequency was higher in TILs than in pPB (P= 0.002), whereas there were no important differences between hPB and pPB. CD25(+) cells isolated either from PB and tumours showed the ability to significantly suppress in vitro both proliferation and interferon-γ production by CD25-depleted MNC, thus demonstrating that they are active Tregs. • Treg frequency was found to significantly correlate both with pathological stage (pPB, P= 0.03; TIL, P= 0.04) and nuclear grade (TIL, P= 0.005), both for UCLA and Kattan models (all: P < 0.05 for both pPB and TIL). CONCLUSION: • Treg frequency is significantly higher in TIL than in pPB of patients with RCC. Tregs showed in vitro an inhibitory activity on effector T cells isolated from kidney tumours. The increase in both peripheral and intratumoral Tregs in subjects affected with RCC were associated with worse prognosis.

Identification of a novel subset of human circulating memory CD4(+) T cells that produce both IL-17A and IL-4.

BACKGROUND: IL-17A has been suggested to play a pathogenic role in bronchial asthma and other allergic disorders. OBJECTIVE: Study of the relationship between human IL-17A-producing CD4(+) T(H) cells (T(H)17) and IL-4-producing CD4(+) T(H) (T(H)2) cells. METHODS: T-cell clones generated from the CCR6(+)CD161(+) fraction of human circulating CD4(+) T cells, which contains virtually all T(H)17 cells, as well as circulating CD4(+) T cells from both healthy subjects and patients with asthma, were assessed by flow cytometry for their cytokine production profile. RESULTS: A small proportion of CCR6(+)CD161(+)CD4(+) T-cell clones showed the ability to produce both IL-17A and IL-4 (T(H)17/T(H)2). T(H)17/T(H)2 clones also produced IL-5, IL-8, IL-9, IL-13, IL-21, and IL-22 and displayed the ability to induce the in vitro secretion of IgE. A very few T(H)17/T(H)2 cells were found among circulating CD4(+) T cells from normal subjects, but their proportions were significantly increased in the circulation of patients with chronic asthma. T(H)17/T(H)2 cells could not be derived from naive umbilical cord blood CD4(+) T cells under any experimental condition. However, when circulating memory CCR6(+)CD161(+)CD4(+) T cells were cloned under appropriate polarizing conditions, T(H)17/T(H)2 clones originated in the presence of IL-4, suggesting that an IL-4-rich microenvironment may induce the shifting of memory T(H)17 cells into T(H)17/T(H)2 cells. CONCLUSION: Because of its peculiar functional properties and the increased numbers in the circulation of patients with bronchial asthma, this previously unknown population of T(H)17/T(H)2 cells may play some role in the pathogenesis of this disease.

TGF-beta indirectly favors the development of human Th17 cells by inhibiting Th1 cells.

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti-proliferative effect of TGF-beta than Th1 and Th2 clones or circulating Th1-oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl-2 expression and reduced apoptosis in the presence of TGF-beta, in comparison with Th1 cells. Umbilical cord blood naïve CD161(+)CD4(+) T cells, which contain the precursors of human Th17 cells, differentiated into IL-17A-producing cells only in response to IL-1beta plus IL-23, even in serum-free cultures. TGF-beta had no effect on constitutive RORgamma t expression by umbilical cord blood CD161(+) T cells but it increased the relative proportions of CD161(+) T cells differentiating into Th17 cells in response to IL-1beta plus IL-23, whereas under the same conditions it inhibited both T-bet expression and Th1 development. These data suggest that TGF-beta is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.

Human immature myeloid dendritic cells trigger a TH2-polarizing program via Jagged-1/Notch interaction.

BACKGROUND: The mechanisms by which human dendritic cells (DCs) activate a TH1-polarizing or TH2-polarizing program are still partially unclear. OBJECTIVE: Study of the mechanisms responsible for the TH1/TH2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs). METHODS: IL-4 and IFN-gamma production by CD4+ T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs. RESULTS: Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4+ T cells a TH2 polarization, as shown by Jagged-1 gene silencing. The TH2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4+ T cells to the TH1 phenotype. CONCLUSION: CD4+ T-cell responses are usually characterized by a prevalent TH2 phenotype unless TLRs are triggered on DCs by microbial components.

Brief report: etanercept inhibits the tumor necrosis factor α-driven shift of Th17 lymphocytes toward a nonclassic Th1 phenotype in juvenile idiopathic arthritis.

OBJECTIVE: To evaluate the effects of etanercept on the phenotype of CD4+ T helper lymphocytes from patients with juvenile idiopathic arthritis (JIA). METHODS: We compared the proportions of various Th cell subsets in peripheral blood (PB) from etanercept-treated and untreated JIA patients. An in vitro study was performed on PB mononuclear cells (PBMCs) from 15 children with untreated JIA, in which we evaluated the proliferative response of these cells, as well as their cytokine production profile, in the presence of various stimuli with or without etanercept. RESULTS: We found lower proportions of CD4+ CD161+ (nonclassic) Th1 lymphocytes in the PB of patients treated with etanercept than in untreated patients. In vitro, etanercept inhibited the proliferative response induced by either polyclonal or recall antigen stimulation of PBMCs. Moreover, etanercept increased the proportion of CD4+CD161+ Th17/Th1 and Th17 cells in vitro while decreasing the proportions of nonclassic Th1 cell subsets, leaving CD4+CD161- (classic) Th1 cells unaffected. We also found that tumor necrosis factor α (TNFα) was able to induce transition of Th17 lymphocytes toward the nonclassic Th1 phenotype in vitro, probably due to the high expression of TNF receptor type II observed in Th17 cells. CONCLUSION: We have previously demonstrated the occurrence of a shifting of CD4+CD161+ Th17 cells to the nonclassic Th1 phenotype in children with JIA. The present findings suggest that etanercept can exert its disease-modifying action by interfering with this shifting.

Human interleukin 17-producing cells originate from a CD161+CD4+ T cell precursor.

We demonstrate that CD161 is a highly up-regulated gene in human interleukin (IL) 17 T helper cell (Th17) clones and that all IL-17-producing cells are contained in the CD161(+) fraction of CD4(+) T cells present in the circulation or in inflamed tissues, although they are not CD1-restricted natural killer T cells. More importantly, we show that all IL-17-producing cells originate from CD161(+) naive CD4(+) T cells of umbilical cord blood, as well as of the postnatal thymus, in response to the combined activity of IL-1 beta and IL-23. These findings implicate CD161 as a novel surface marker for human Th17 cells and demonstrate the exclusive origin of these cells from a CD161(+)CD4(+) T cell progenitor.